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. 2011 Feb 18;286(7):5175-86.
doi: 10.1074/jbc.M110.187872. Epub 2010 Nov 30.

Palmitoylation controls dopamine transporter kinetics, degradation, and protein kinase C-dependent regulation

James D Foster  1 , Roxanne A Vaughan
Affiliations

Palmitoylation controls dopamine transporter kinetics, degradation, and protein kinase C-dependent regulation

James D Foster et al. J Biol Chem. .

Abstract

Palmitoylation is a lipid modification that confers diverse functions to target proteins and is a contributing factor for many neuronal diseases. In this study, we demonstrate using [(3)H]palmitic acid labeling and acyl-biotinyl exchange that native and expressed dopamine transporters (DATs) are palmitoylated, and using the palmitoyl acyltransferase inhibitor 2-bromopalmitate (2BP), we identify several associated functions. Treatment of rat striatal synaptosomes with 2BP using lower doses or shorter times caused robust inhibition of transport V(max) that occurred with no losses of DAT protein or changes in DAT surface levels, indicating that acute loss of palmitoylation leads to reduction of transport kinetics. Treatment of synaptosomes or cells with 2BP using higher doses or longer times resulted in DAT protein losses and production of transporter fragments, implicating palmitoylation in regulation of transporter degradation. Site-directed mutagenesis indicated that palmitoylation of rat DAT occurs at Cys-580 at the intracellular end of transmembrane domain 12 and at one or more additional unidentified site(s). Cys-580 mutation also led to production of transporter degradation fragments and to increased phorbol ester-induced down-regulation, further supporting palmitoylation in opposing DAT turnover and in opposing protein kinase C-mediated regulation. These results identify S-palmitoylation as a major regulator of DAT properties that could significantly impact acute and long term dopamine transport capacity.

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Figures

FIGURE 1.
FIGURE 1.
S-Palmitoylation of DAT. rDAT-LLCPK1 cells (A) and rat striatal synaptosomes (B) were metabolically labeled with [3H]palmitic acid for 18 h or 90 min, respectively, and lysates were precipitated with Ab16 followed by SDS-PAGE/fluorography. Preimmune antiserum (pre) and LLCPK1 cells served as negative controls, and migration of bands was verified by parallel electrophoresis of [125I]RTI 82-labeled DAT. For acyl thioester analyses, precipitates from cells or synaptosomes were treated with Tris-HCl or hydroxylamine (NH2OH) as indicated. C, rDAT-LLCPK1 cells were incubated with [3H]palmitate for the indicated times followed by immunoprecipitation and SDS-PAGE/fluorography of DAT. D, mouse striatal synaptosomes and His6-hDAT HEK 293 cells were labeled with [3H]palmitate for 90 min or 18 h, respectively, and DATs were precipitated with Ab16 or anti-His antibodies followed by SDS-PAGE/fluorography. Results are representative of three or more independent experiments.
FIGURE 2.
FIGURE 2.
DAT palmitoylation specificity and inhibition by 2BP. A, rDAT-LLCPK1 cells or His6-hDAT-HEK 293 cells were labeled for 18 h with 10 μm [3H]palmitate in the presence or absence of 15 μm 2BP. B, rat striatal synaptosomes were labeled with 40 μm [3H]palmitic acid or 40 μm [3H]myristic acid (Myr) for 90 min. Equal amounts of DAT were immunoprecipitated and analyzed by SDS-PAGE/fluorography ([3H]pal) or immunoblotting (IB). C, rat striatal synaptosomes were treated with 5 μm 2BP for 45 min at 30 °C followed by acyl-biotinyl exchange (ABE). Samples were treated with Tris-HCl, pH 7.4 (control), or NH2OH to remove thioester-linked palmitate, followed by sulfhydryl-reactive biotinylation, NeutrAvidin extraction, and DAT immunoblotting. D, DAT immunoblot of vehicle and 2BP-treated samples. All results are representative of three or more independent experiments.
FIGURE 3.
FIGURE 3.
2BP reduces DA transport activity and DAT levels in rat striatal synaptosomes. Rat striatal synaptosomes were treated with vehicle (no treatment) or the indicated concentrations of palmitate (Pal) or 2BP at 30 °C for 60 min, and aliquots were analyzed for [3H]DA transport, [3H]alanine transport, or DAT immunoblotting. A, [3H]DA transport activity (means ± S.E. of 3–4 experiments performed in triplicate) relative to vehicle controls normalized to 100%. *, p < 0.05; ***, p < 0.001, 2BP or Pal versus vehicle; ††, p < 0.01, †††, p < 0.001 2BP versus Pal (one-way ANOVA with Tukey's post test). B, equal amounts of protein from treated synaptosomes (conditions indicated directly below on histogram) were immunoblotted for DAT (representative blot shown), and band density was expressed as a fraction of control values normalized to 100% (means ± S.E. of three experiments performed in duplicate). *, p < 0.05, 2BP versus vehicle control (one-way ANOVA with Tukey's post test). DAT blots were stripped and re-probed for α-tubulin, which showed no change with Pal or 2BP treatment. C, [3H]alanine transport activity (means ± S.E. of three experiments performed in triplicate) relative to controls normalized to 100%.
FIGURE 4.
FIGURE 4.
Kinetic analysis of 2BP effects in synaptosomes. A, rat striatal synaptosomes were treated for the indicated times with vehicle or 5 μm 2BP at 30 °C followed by [3H]DA transport assay. Transport values obtained with 2BP treatment are calculated as the fraction of activity relative to each time point control. Values shown are means ± S.E. of three experiments performed in triplicate; *, p < 0.05; ***, p < 0.001 relative to time point control (one-way ANOVA with Tukey's post test). B, saturation analysis of rat striatal synaptosomes treated with vehicle or 5 μm 2BP for 45 min. Results shown are means ± S.E. of three independent experiments performed in triplicate.
FIGURE 5.
FIGURE 5.
Surface biotinylation analysis of 2BP-treated synaptosomes. Percoll gradient-purified synaptosomes were treated with vehicle, 1 μm PMA, or 7.5 μm 2BP for 30 min at 30 °C, and aliquots were assayed for [3H]DA transport activity or subjected to surface biotinylation. All results shown are means ± S.E. of three independent experiments performed in triplicate. A, [3H]DA transport activity of synaptosomes normalized to vehicle controls; *, p < 0.05; ***, p < 0.001; relative to control by ANOVA with Dunnett's post test. B, equal amounts of protein from control and treated synaptosomes were immunoblotted for DAT or tyrosine hydroxylase (TH). Upper panel shows representative immunoblots (lanes correspond to treatments shown directly below on histogram). Histogram shows quantification of DAT levels, **, p < 0.01 relative to control by ANOVA with Dunnett's post test. C, fractions eluted from NeutrAvidin resin were immunoblotted for DAT and tyrosine hydroxylase (upper panels). Histogram shows quantification of biotinylated DAT normalized for total DAT protein, p > 0.05 by ANOVA with Dunnett's post test.
FIGURE 6.
FIGURE 6.
Reduction of rDAT palmitoylation by C580A mutation. A, diagram of rDAT modeled as in Beuming et al. (49) highlighting Cys residues analyzed in this study (numbered circles). B, LLCPK1 cells stably expressing WT or Cys → Ala rDATs were labeled with [3H]palmitate, and equal amounts of DAT were immunoprecipitated with Ab16 followed by SDS-PAGE/fluorography (top panel) or were immunoblotted (middle panel). LLCPK1 cells were labeled in parallel, and lysate amounts equal to that used for WT DAT were analyzed as negative controls. Bottom panel, equal amounts of WT and C580A DAT were precipitated with immune (16) or preimmune (pre) antiserum followed by SDS-PAGE/auto fluorography. IB, immunoblot. C, quantification of DAT [3H]palmitate labeling relative to DAT protein levels (means ± S.E. of 2–3 experiments as indicated performed in duplicate). *, p < 0.05 relative to WT, student's t test.
FIGURE 7.
FIGURE 7.
2BP effects on DA transport and DAT levels in heterologous cells. A, rDAT-LLCPK1 cells were treated with vehicle or 15 μm 2BP for 18 h followed by [3H]DA transport assay or immunoblotting of equal amounts of protein for DAT. Left, histogram shows transport activity (means ± S.E. of four independent experiments performed in triplicate). ***, p < 0.001, 2BP versus control (one-way ANOVA with Dunnett's post test). Right, representative immunoblot of DAT from control and 2BP-treated cells. B, saturation analysis rDAT-LLCPK1 cells treated with or without 15 μm 2BP for 18 h. Results shown are means ± S.E. of six independent experiments.
FIGURE 8.
FIGURE 8.
Enhancement of DAT losses and degradation fragment production with C580A mutation and 2BP treatment. A, equal amounts of DAT from WT, C522A, or C580A rDAT-LLCPK1 cell lysates were immunoblotted with MAb16. Markers indicate mature glycosylated protein (M), immature nonglycosylated protein (IM), and fragments (F). B, equal amounts of protein from C580A DAT-LLCPK1 cells treated with vehicle (Veh) or 15 μm 2BP for 18 h were immunoblotted for DAT using control or peptide 16 preabsorbed antiserum.
FIGURE 9.
FIGURE 9.
2BP and PMA effects on WT and C580A DAT levels. A, cells expressing WT or C580A DATs were treated with vehicle or 15 μm 2BP for 18 h followed by vehicle or 1 μm PMA for the final 30 min. Lysates were immunoblotted for DAT, and markers indicate mature (M) and immature (IM) DAT forms and fragments (F). B, quantification of full-length DAT from each treatment (means ± S.E. of four independent experiments). *, p < 0.05; ***, p < 0.001, indicated treatments versus control; †, p < 0.05; †††, p < 0.001, C580A versus WT for indicated treatments (one-way ANOVA with Tukey's post test).
FIGURE 10.
FIGURE 10.
Additivity of PMA, 2BP, and C580A effects on DA transport activity. A, rat striatal synaptosomes were treated with the indicated combinations of 5 μm 2BP and 1 μm PMA for 30 min followed by assay for [3H]DA transport. Results shown are means ± S.E. of four independent experiments performed in triplicate. ***, p < 0.001 indicated treatments versus control; ##, p < 0.05; ###, p < 0.001; 2BP plus PMA versus 2BP or PMA only (one-way ANOVA with Tukey's post hoc test). Ctl, control. B, cells expressing WT or C580A DATs were treated with vehicle or 15 μm 2BP for 18 h followed by vehicle or 1 μm PMA for the final 30 min prior to [3H]DA transport assay. Results shown are means ± S.E. of 3–5 independent experiments performed in triplicate. **, p < 0.01; ***, p < 0.001 indicated treatments versus control; #, p < 0.05; ##, p < 0.01; ###, p < 0.001, WT or C580A DATs, PMA plus 2BP versus PMA, or 2BP only; †, p < 0.05; ††, p < 0.01, C580A versus WT DATs for indicated treatment groups (one-way ANOVA with Tukey's post hoc test).
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